Therapeutic Antibodies & Biosimilars: Comparative Analysis

Therapeutic Antibodies

Biologics are generally large molecules, mainly proteins, produced by living cells. Their structures are considerably more complex than those of small-molecule drugs. Monoclonal therapeutic antibodies constitute the largest group of biologics. Thus, in an analysis of the contribution of X-ray crystallography to drug discovery and design for the period 2010-2016, Westbrook & Burley (2019) identified 210 PDB entries describing drugs newly approved by the FDA. Of those, 36 were biologics, of which 24 were monoclonal antibodies. The development of new antibody therapies has become a significant focus of modern drug design. The information on antibody three-dimensional structure, combined with antibody engineering, plays a central role in this process and has opened the way for the rapid growth of new antibody therapies. Currently (2025), according to the Antibody Therapies Database (UmabsDB), there are over 11,000 monoclonal antibodies targeting over 2,400 human disease conditions, including psoriasis, Crohn’s disease, ulcerative colitis, arthritis, kidney conditions, diabetes, and cancers (such as breast, lung, and colon).

The first monoclonal therapeutic antibody targeting CD3, a cell-surface protein complex with a critical role in T-cell biology, was approved by the FDA in 1986. The antibody was developed by Stuart Schlossman and colleagues and later named muromonab-CD3. It was introduced into the clinic as an immunosuppressant for the prevention of transplant rejection. Thirty-five years later, in 2021, the FDA approved the 100th therapeutic antibody. In Europe, the first monoclonal antibody therapy was approved in 1991 (withdrawn in 1993), and the second in 1995. Japan and China have been lagging after the USA and Europe, but are rapidly catching up. The number of approved therapeutic antibodies in different countries is also reflected in the number of companies involved in this business. As of 2025, there are 229 therapeutic antibodies (bisimilars not included) approved in the USA or EU.

Therapeutic Antibodies & Biosimilars: Comparability Analysis

Considering the time interval since the first therapeutic antibody approval in 1986, we may expect several antibodies already have or are coming out of patent in the next few years. And as with small-molecule drugs, pharmaceutical companies are racing to develop their follow-on variants, called biosimilars. Currently (Nov 2025), 144 biosimilars have been approved by EMA for use in the EU. The FDA has approved 77 biosimilars.

Unlike generic small-molecule drugs, which are synthesized in the laboratory and are exact copies of the reference drug, therapeutic antibodies are produced in mammalian cells due to the ability of these cells to perform post-translational modification. A drawback of expression in heterologous systems is unpredictable modifications like variable glycosylation, deamination of asparagine and glutamine, and oxidation of cysteine, methionine, or tryptophan, which result in heterogeneity in the biologic product. Unexpected modifications may affect the product’s chemical properties, such as surface charge, solubility, stability, and three-dimensional structure (higher order structure or HOS). These, in turn, may affect the efficacy and safety of the therapeutic antibody treatment. For this reason, biosimilars cannot be handled as generic small-molecule drugs (and they are not considered “generic drugs”). For approval by regulatory agencies, developers must thus present detailed comparative studies of the biosimilar, which has to be highly similar to the reference therapeutic antibody, with no clinical differences in safety, quality, and efficacy, as stated by EMA. Among these parameters, biosimilar structure comparability analysis plays a central role.

The American FDA recommends using a stepwise process to prove biosimilarity “without the need to establish the safety and effectiveness of the proposed product independently.” The process is divided into 1) biosimilar structural analysis, 2) functional analysis, 3) animal data, and 4) clinical studies. First, extensive structural and functional characterization and comparison of the proposed and reference products should be performed. The guidelines stress, “The more comprehensive and robust the comparative structural and functional characterization, the more useful such characterization will be in determining what additional studies may be needed.” In addition, the FDA stresses that “appropriate analytical methodologies with adequate sensitivity and specificity for structural characterization of the proteins should be used.”

Therapeutic antibody and biosimilar higher order structure (HOS) analysis should compare the primary (amino acid sequence) and higher order structures, including secondary, tertiary, and quaternary structures. The EMA stresses the importance of comparability of the biosimilar’s and the originator therapeutic antibody’s HOS. Undoubtedly, detailed structural characterization is highly relevant to the process. This is understandable, given the importance of the molecule’s overall conformation, especially the conformation of the compatibility-determining and framework regions (CDR and FR), which are essential for antigen recognition. Alterations in these structures may affect the therapeutic antibody’s ability to recognize its antigen and reduce its efficacy.

Methods like UV and fluorescent spectroscopy are recommended for tertiary structure analysis, while Fourier Transform Infrared (FTIR) and CD spectroscopy are recommended for secondary structure analysis. However, as recent research has demonstrated, 2D NMR spectroscopy yields much more accurate and reliable information. These results are discussed in detail in our article on higher order structure analysis. The SARomics Biostructures’ team uses 2D NMR spectroscopy alone or in combination with X-ray crystallography for cost-efficient and high-precision comparability analysis of therapeutic antibody and biosimilar higher order structure.

Follow us on LinkedIn to get updates on our future articles. For project discussions, please see our antibody structure services, NMR spectroscopy services, and protein X-ray crystallography services pages or contact us directly. We also invite you to visit our publications page for examples of therapeutic antibody projects we have worked on.